doi: 10.1128/MCB.23.23.8528-8541.2003.
Positive and negative regulation of the cardiovascular transcription factor KLF5 by p300 and the oncogenic regulator SET through interaction and acetylation on the DNA-binding domain
Affiliations
- PMID:14612398
- PMCID: PMC262669
- DOI: 10.1128/MCB.23.23.8528-8541.2003
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Positive and negative regulation of the cardiovascular transcription factor KLF5 by p300 and the oncogenic regulator SET through interaction and acetylation on the DNA-binding domain
Saku Miyamoto et al. Mol Cell Biol.2003 Dec.
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Abstract
Here we show a novel pathway of transcriptional regulation of a DNA-binding transcription factor by coupled interaction and modification (e.g., acetylation) through the DNA-binding domain (DBD). The oncogenic regulator SET was isolated by affinity purification of factors interacting with the DBD of the cardiovascular transcription factor KLF5. SET negatively regulated KLF5 DNA binding, transactivation, and cell-proliferative activities. Down-regulation of the negative regulator SET was seen in response to KLF5-mediated gene activation. The coactivator/acetylase p300, on the other hand, interacted with and acetylated KLF5 DBD, and activated its transcription. Interestingly, SET inhibited KLF5 acetylation, and a nonacetylated mutant of KLF5 showed reduced transcriptional activation and cell growth complementary to the actions of SET. These findings suggest a new pathway for regulation of a DNA-binding transcription factor on the DBD through interaction and coupled acetylation by two opposing regulatory factors of a coactivator/acetylase and a negative cofactor harboring activity to inhibit acetylation.
Figures
Isolation of interactors of KLF5. (A) Silver-stained gel of the histidine-tagged KLF5 ZF/DBD recombinant used for interaction studies. Molecular mass markers are shown on left. (B) Isolation of factors associating with KLF5 ZF/DBD. Histidine-tagged KLF5 ZF/DBD was bound to nickel-chelating resin and subjected to VSMC C2/2 nuclear extract (NE) (lane 2). Lane 1 is nuclear extract alone, and lane 3 is recombinant protein immobilized on resin alone. Eluate was resolved by SDS-PAGE (12% polyacrylamide) and stained by Coomassie brilliant blue. The stained band, which was excised and subjected to further analysis, is indicated p39. (C) MALDI-TOF mass spectra obtained from tryptic peptides of p39. Fragment peaks assigned to SET are marked (asterisks). Peaks indicated A and B were subjected to postsource decay sequencing. (D) Partial peptide sequences of human SET. Numbering is from the initiation methionine of SET. The peptide sequences of peaks A and B obtained by postsource decay are boxed. (E) In vitro binding of KLF5 ZF/DBD and SET. Immobilized GST-KLF5 ZF/DBD fusion protein was reacted with histidine-tagged SET protein, separated by SDS-PAGE, and analyzed by immunoblotting with anti-HIS probe antibody (lane 3). Lane 1 is the input. GST protein was used as the control (lane 2). (F) In vitro binding of KLF5 full-length protein and SET. Immobilized GST-KLF5 full-length fusion protein was reacted with histidine-tagged SET protein, separated by SDS-PAGE, and analyzed by immunoblotting with anti-HIS probe antibody (lane 3). Lane 1 is the input. GST protein was used as a control (lane 2). (G) Coimmunoprecipitation of SET with KLF5. Cell lysate was immunoprecipitated with anti-KLF5 rat monoclonal antibody (lane 2) or normal rat IgG (lane 1) as a control. Bound materials were separated by SDS-PAGE and analyzed by immunoblotting with anti-SET antibody. (H) Intracellular localization of KLF5 and SET. Endogenous SET (a) (green) and KLF5 (b) (red) were detected mainly in nuclei. Confocal microscopy double-staining analysis indicates colocalization of SET and KLF5 (c) (yellow). All experiments were done at least twice with consistent findings. +, present; −, absent.
Effects of SET on KLF5 activity. (A) Effects of SET on KLF5 DNA-binding activity. A gel shift assay with recombinant KLF5 ZF/DBD and SET was performed. Wt and mut represent wild and mutant oligonucleotide competitors (lanes 5 and 6). The amount of recombinant protein is as follows: 150 (+) and 450 (++) ng for SET (lanes 2, 7, and 8) and 10 (+) and 50 (++) ng for KLF5 ZF/DBD (lanes 3 to 8). (B) Gel shift assay of control NF-κB p50 subunit and SET. Gel shift units (0.1) of NF-κB p50 (lanes 2, 4, and 5) and 100 (+) and 300 (++) ng of SET (lanes 3, 4, and 5) were used. (C) Effects of SET on KLF5 transactivation. Cotransfection analysis of effects of SET on KLF5 transcriptional activation.One hundred nanograms of reporter was used in each lane. Effectors are as follows: lanes 2, 3, and 4 through 7, and 12, 13, and 14 through 17 were 83, 250, and 750 ng of KLF5 expression plasmid (pCAG-KLF5), respectively; lanes 5 and 8, 6 and 9, 7 and 10, 15 and 18, 16 and 19, and 17 and 20 were 28, 83, and 250 ng of SET expression plasmid (pCHA-SET/TAF-Iβ). The total amount of effector plasmid was adjusted to 1 μg with the respective control vector. (D) Effects of SET on control NF-κB transactivation. NF-κB transactivation (lanes 2 to 5) was done by transfection of equal amounts (250 ng) of p50 and p65 subunit expression vectors. (E) Effects of SET on KLF5-induced cell growth. SET was transiently transfected into cells stably expressing epitope-tagged (3× FLAG) KLF5 or mock vector in 3T3-3 cells. The cell count on day 5 after transfection compared with mock vector-treated cells is shown. Error bars denote standard errors. (F) BrdU assay showing effects of KLF5 and SET on cell growth by use of adenovirus-mediated transfer (multiplicity of infection, 100) of KLF5 and SET adenoviruses. Empty (lane 1) denotes empty vector alone. Error bars denote standard errors. OD450, optical density at 450 nm. (G) In vitro binding of KLF5 ZF/DBD and SET deletion mutants. Immobilized histidine-tagged SET protein was reacted with GST-KLF5 ZF/DBD fusion protein, separated by SDS-PAGE, and analyzed by immunoblotting with anti-GST antibody. SET deletion mutants are shown by their amino acid numbers in reference to the schematic diagram of functional domains shown above. Lane 1 is GST KLF5 ZF/DBD input, and lane 2 shows that GST KLF5 ZF/DBD does not bind Probond nickel-chelating resin. Lanes 3 to 8 show GST KLF5 ZF/DBD binding to respective resin-bound deletion mutants. Amino acids (aa) 1 to 24 comprise the SET-specific N-terminal region, amino acids 25 to 65 comprise the coiled-coil dimerization domain, amino acids 25 to 119 comprise a region known to inhibit phosphatase PP2A, amino acids 120 to 225 comprise a region with unknown function, and amino acids 226 to 277 comprise the acidic C-terminal region. All experiments were done at least twice with consistent findings.
Effects of SET on KLF5 downstream gene expression and pathological states. (A) Induction of KLF5 protein and repression of SET protein after mitogenic stimulation. Cells were starved at the times shown for 24 h, incubated with 100 ng of PMA/ml for the indicated times, and then harvested. Cell lysate was resolved by SDS-PAGE and subjected to Western blotting or Coomassie brilliant blue staining. (B) Quantification of KLF5 and SET protein levels. KLF5 and SET protein levels were normalized by the corresponding Coomassie brilliant blue staining pattern. The relative expression level was shown as the level at 0 h. (C) Induction of PDGF-A chain mRNA expression. Cells were starved at the times shown for 24 h, incubated with 100 ng of PMA/ml for the indicated times, and then harvested. The quantitative reverse transcription-PCR fragmentwas resolved on a 2% agarose gel. (D) Quantification of mRNA expression level for the PDGF-A chain. The expression level of the PDGF-A chain, an endogenous target gene of KLF5, was normalized to that of 18S. (E) KLF5 and SET expression in the pathological neointima. The immunohistochemistry of SET and KLF5 in a balloon injury model of atherosclerosis was examined. The left common carotid artery was denudated by balloon injury, and the neointima was observed 2 weeks after the balloon injury (Injured). The right common carotid artery served as a control (Control). The rat aorta was stained with anti-SET and anti-KLF5 antibodies. Cells in the neointima were clearly positive for SET and KLF5. All experiments were done at least twice with consistent findings. H/E, hematoxylin and eosin staining.
Acetylation of KLF5 and its regulation by SET. (A) Acetylation of KLF5 in vivo. Cells were treated with trichostatin A and labeled with [3H]acetate followed by immunoprecipitation with KLF5 (lane 2) and control normal IgG antibodies (lane 1). (B) Schematic representation of GST-KLF5 fusion mutant constructs. GST-KLF5 comprises full-length KLF5 fused to GST, GST-KLF5-ΔZF/DBD comprises only the N-terminal regulatory domain fused to GST, and GST-KLF5-ZF/DBD comprises only the C-terminal zinc finger DBD fused to GST. (C) Acetylation of KLF5 mutant constructs in vitro by p300. KLF5 proteins (1.2 μg) were incubated with 50 ng of FLAG-p300 HAT domain protein (amino acids 1195 to 1673) in the presence of [14C]acetyl-CoA. Reaction products were separated by SDS-12% PAGE. The difference between pairs (lanes 1 and 2, 3 and 4, and 5 and 6) is the presence of p300 HAT protein in the reaction mixture for the respective KLF5 mutant proteins. The gel was stained with Coomassie brilliant blue (lower panel) and then analyzed with a BAS 1500 phosphorimager (upper panel). (D) Effects of acetylation on KLF5 DNA-binding activity. Acetylation reactions were performed in the presence (+) of acetyl-CoA (AcCoA) and FLAG-p300 HAT domain (lane 3), in the presence of FLAG-p300 HAT domain (lane 2), and in the absence (−) of acetyl-CoA or FLAG-p300 HAT domain (lane 1). Reaction products were resolved by electrophoresis and analyzed with BAS1500. (E) Effects of SET on KLF5 acetylation (lanes 5 to 8). Histone H4 was used as a control (lanes 1 to 4). A schematic diagram of the protocol for order-of-addition experiments is shown. In lanes 3 and 7, the p300 HAT domain was added following the reaction of SET with the substrate (KLF5 ZF/DBD or histone H4) (prior to acetylation), and in lanes 4 and 8, SET was added following the reaction of p300 HAT with the substrate (after acetylation). Acetylation reactions were done essentially as described above. All experiments were done at least twice with consistent findings.
Interaction and activation of KLF5 by the coactivator and acetylase p300. (A) Interaction of KLF5 and p300 in vivo. p300 was immunoprecipitated from cells followed by immunoblotting against KLF5 (lane 2). Immunoblotting against p300 confirms the immunoprecipitation procedure (lane 4). Normal IgG was used as the control (lane 2). (B) Interaction between KLF5 deletion mutants and p300 HAT domain in vitro. Approximately 2 μg of recombinant GST-KLF5 wild type (wt) (amino acids 1 to 457, lane 3), GST-KLF5-ΔDBD (amino acids 1 to 367, lane 4), and GST-KLF5-DBD (amino acids 368 to 457, lane 5) were used in a GST pull-down assay with 1 μg of the FLAG-p300 HAT domain. The interaction between KLF5 and the p300 HAT domain was analyzed by SDS-10% PAGE of the pull-down reactions and Western blotting with anti-FLAG antibody. The input (lane 1) contains 10% of the FLAG-p300 HAT domain protein. GST protein (lane 2) was used as a control. (C) Effects of p300 on KLF5 transactivation as assessed by reporter cotransfection assay. Cells were transfected with 100 ng of PDGF-A chain luciferase reporter and increasing amounts of KLF5 expression vector plasmids up to 750 ng (lanes 2 to 7). DNA concentrations were maintained constant by ad-dition of the empty vector. Increasing amounts of p300 expression vector plasmids were similarly cotransfected up to 250 ng with 100 ng of PDGF-A-luciferase reporter plasmid in the absence (−) (lanes 8 and 9) or presence of 750 ng of KLF5 expression vector (lanes 4 and 5). Effects of an acetyltransferase region-deleted mutant of p300 (ΔHAT) on KLF5-mediated transcriptional activation were also examined (lanes 6, 7, 10, and 11). All experiments were done at least twice with consistent findings. +, present.
Mapping of the acetylated region and residue of KLF5. (A) Schematic representation of KLF5 zinc finger peptides. ZF1, ZF2, and ZF3 cover each of the zinc fingers, respectively, from the N terminus. (B) Acetylation of KLF5 zinc finger mutants in vitro. Approximately 1.0 μg of purified GST-KLF5 fusion zinc fingers 1, 2, and 3 were incubated with [14C]acetyl-CoA and recombinant FLAG-p300 HAT domain. Reaction products were separated by SDS-10% PAGE. The gel was stained with Coomassie brilliant blue (lower panel) and then analyzed with a BAS 1500 phosphorimager (upper panel). (C) Mass spectrum quantification of acetylated lysines in peptide KLF5-ZF1. A parallel reaction mixture with unlabeled acetyl-CoA was analyzed by MALDI-TOF (MS). The major peak labeled X corresponds to the expected mass of the unmodified peptide KLF5-ZF1. The major peak labeled Y, larger by 42 atomic mass units, represents monoacetylated peptide. (D) Masses of peptides digested withLys-C endopeptidase. The peptide sequences that are suggested from measured masses are shown below. Peak X represents the acetylated fragment. (E) Replacement of acetylated lysine by arginine impairs acetylation of GST-KLF5-zinc finger 1. Approximately 1.0 μg of purified GST-KLF5-zinc finger 1 (lane 1, wild type [wt]) and GST-KLF5-mut zinc finger 1 (K369R) (lane 2, mutant [mut]) were incubated with [14C]acetyl-CoA and 50 ng of FLAG-p300 HAT domain protein. Reaction products were separated by SDS-10% PAGE. The gel was stained with Coomassie brilliant blue (lower panel) and then analyzed with a BAS 1500 phosphorimager (upper panel). (F) Binding assay of K369R and wild-type KLF5 with SET. Wild-type and K369R mutant KLF5 fused to GST were immobilized on GST resin followed by a pull-down assay of SET protein. Lanes 1 and 4 are SET input. Lanes 2 and 5 are GST alone. All experiments were done at least twice with consistent findings. +, present; −, absent.
Effects of the KLF5 K369R point mutant. (A) Effects of KLF5 K369R mutant (lane 3) on PDGF-A chain promoter transcriptional activation compared to that of the KLF5 wild type (wt) (lane 2). Seven hundred fifty nanograms of each expression vector was transfected in the presence of 100 ng of the reporter construct (all lanes). −, absent. (B) Effects of SET on KLF5 wild type and K369R mutant. Up to 250 ng of SET expression vector was transfected in the presence of 750 ng of KLF5 expression vector. (C) Effects of the KLF5 K369R mutant on PDGF-A chain promoter transcriptional activation compared to that of the KLF5 wild type in the presence of p300. Increasing amounts of p300 expression vector up to 250 ng were transfected in the presence of 750 ng of KLF5 (wild type, lanes 1 to 4; K369R mutant, lanes 5 to 8) expression vector and 100 ng of the reporter construct. (D) Effects of KLF5 K369R mutant on cell growth. The KLF5 wild type and K369R mutant were transfected with adenovirus and counted on the sixth day in comparison with nontreated cells. Error bars denote standard errors. (E) Effects of the KLF5 K369R mutant on cell growth were similarly assessed by BrdU assay. Error bars denote standard errors. All experiments were done at least twice with consistent findings. (F) Summary of findings. Note that SET negatively regulates DNA binding, transactivation, and acetylation of KLF5. p300 interacts, transactivates, and acetylates KLF5. We envision that this mechanism of interplay between coupled positive regulation by p300 and negative regulation by SET occurs in an inducible setting (e.g., phorbol ester stimulation).
References
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